Our objective is to engineer the release of adjuvant co-entrapped with tumor specific antigens for immunotherapeutic treatment of melanoma. The specific aims are: (1) Engineer biodegradable nanoparticles for enhanced immunotherapeutic responses by preparing cytosine-phosphorothiolate-guanine oligodeoxynucleotides (CpG)-antigen fusion conjugates and double layered particles with antigens covalently attached on the surface of the particles and CpG entrapped within the particle matrix. Following optimization of the particle formulation methodology, the immune response initiated by the particle vaccines will be characterized by quantifying the cytokine/chemokine production and upregulation and downregulation of Toll-like receptor 9 (TLR9), IFN-3, IL- 6 and IL-12 using RT-PCR and ELISA assays. (2) Evaluate the optimal pre-clinical vaccine strategy for immunotherapeutic treatment in a murine melanoma model by determining the dosing regimen (route of administration and number of doses) that produces maximal immune response. The optimized nanoparticle vaccine will then be tested against alternative immunotherapeutic treatments. The proposed research represents a novel approach of nanoparticle engineering to provide the optimal release kinetics of adjuvant and antigens in stimulating potent immunotherapeutic responses against carcinomas. The development of a potent immunotherapeutic vaccine against melanoma will establish a framework which can be applied to a range of other cancer models including lymphoma, renal carcinoma, colon cancer and pancreatic cancer. PUBLIC HEALTH RELEVANCE: Our objective is to engineer the release of adjuvant co-entrapped with tumor specific antigens for immunotherapeutic treatment of melanoma. The specific aims are: (1) Engineer biodegradable nanoparticles for enhanced immunotherapeutic responses by preparing cytosine-phosphorothiolate-guanine oligodeoxynucleotides (CpG)-antigen fusion conjugates and double layered particles with antigens covalently attached on the surface of the particles and CpG entrapped within the particle matrix. (2) Evaluate the optimal pre-clinical vaccine strategy for immunotherapeutic treatment in a murine melanoma model by determining the dosing regimen (route of administration and number of doses) that produces maximal immune response.